LOCUS       sequence-250081-        4481 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes the BxBI attB GFP dropout destination vector as a type 0 
            part to be used in the MTK system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4481)
  AUTHORS   Fonseca JP, Bonny AR, Kumar GR, Ng AH, Town J, Wu QC, Aslankoohi E, 
            Chen SY, Dods G, Harrigan P, Osimiri LC, Kistler AL, El-Samad H
  TITLE     A Toolkit for Rapid Modular Construction of Biological Circuits in 
            Mammalian Cells.
  JOURNAL   ACS Synth Biol. 2019 Nov 15;8(11):2593-2606. doi: 
            10.1021/acssynbio.9b00322. Epub 2019 Nov 5.
  PUBMED    31686495
REFERENCE   2  (bases 1 to 4481)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1021/acssynbio.9b00322"; journalName: "ACS Synth Biol"; date: 
            "2019-11-15- 15"; volume: "8"; issue: "11"; pages: "2593-2606"
COMMENT     Sequence Label: MTK0_014
FEATURES             Location/Qualifiers
     source          1..4481
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     218..237
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    429..462
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     RBS             788..799
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             806..1519
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      1531..1602
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1618..1645
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     protein_bind    2446..2483
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     CDS             2492..2890
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     primer_bind     complement(2902..2919)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2908..3115
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             complement(3170..3985)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(3170..3985)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3893..3912
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3977..3996
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      join(4210..4481,1..317)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
       61 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
      121 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
      181 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
      241 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
      301 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
      361 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtgcgg
      421 ccgccaatat aacttcgtat aatgtatgct atacgaagtt atccctgaat tcgcatctag
      481 actgatgaga cgtggtagag ccacaaacag ccggtacaag caacgatctc caggaccatc
      541 tgaatcatgc gcggatgaca cgaactcacg acggcgatca cagacattaa cccacagtac
      601 agacactgcg acaacgtggc aattcgtcgc aatacaacgg aaagtgaaac gtgatttcat
      661 gcgtcatttt gaacattttg taaatcttat ttaataatgt gtgcggcaat tcacatttaa
      721 tttatgaatg ttttcttaac atcgcggcaa ctcaagaaac ggcaggttcg gatcttagct
      781 actagagaaa gaggagaaat actagatgcg taaaggcgaa gagctgttca ctggtgtcgt
      841 ccctattctg gtggaactgg atggtgatgt caacggtcat aagttttccg tgcgtggcga
      901 gggtgaaggt gacgcaacta atggtaaact gacgctgaag ttcatctgta ctactggtaa
      961 actgccggtt ccttggccga ctctggtaac gacgctgact tatggtgttc agtgctttgc
     1021 tcgttatccg gaccatatga agcagcatga cttcttcaag tccgccatgc cggaaggcta
     1081 tgtgcaggaa cgcacgattt cctttaagga tgacggcacg tacaaaacgc gtgcggaagt
     1141 gaaatttgaa ggcgataccc tggtaaaccg cattgagctg aaaggcattg actttaaaga
     1201 ggacggcaat atcctgggcc ataagctgga atacaatttt aacagccaca atgtttacat
     1261 caccgccgat aaacaaaaaa atggcattaa agcgaatttt aaaattcgcc acaacgtgga
     1321 ggatggcagc gtgcagctgg ctgatcacta ccagcaaaac actccaatcg gtgatggtcc
     1381 tgttctgctg ccagacaatc actatctgag cacgcaaagc gttctgtcta aagatccgaa
     1441 cgagaaacgc gatcatatgg ttctgctgga gttcgtaacc gcagcgggca tcacgcatgg
     1501 tatggatgaa ctgtacaaat gaccaggcat caaataaaac gaaaggctca gtcgaaagac
     1561 tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tctactagag tcacactggc
     1621 tcaccttcgg gtgggccttt ctgcgtttat agctggaaat ctgctcgtca gtggtgctca
     1681 cactgacgaa tcatgtacag atcataccga tgactgcctg gcgactcaca actaagcaag
     1741 acagccggaa ccagcgccgg cgaacaccac tgcatatatg gcatatcaca acagtccacg
     1801 tctcaagcag ttacagagat gttacgaacc actagtgcac tgcagatcga attcgagttg
     1861 gcgcgcctgt cattctaaat ctctctttca gcctaaagct ttttccccgt atccccccag
     1921 gtgtctgcag gctcaaagag cagcgagaag cgttcagagg aaagcgatcc cgtgccacct
     1981 tccccgtgcc cgggctgtcc ccgcacgctg ccggctcggg gatgcggggg gagcgccgga
     2041 ccggagcgga gccccgggcg gctcgctgct gccccctagc gggggaggga cgtaattaca
     2101 tccctggggg ctttgggagg gggctgtccc cgtgagctcc caggcgcgcc tgtcattcta
     2161 aatctctctt tcagcctaaa gctttttccc cgtatccccc caggtgtctg caggctcaaa
     2221 gagcagcgag aagcgttcag aggaaagcga tcccgtgcca ccttccccgt gcccgggctg
     2281 tccccgcacg ctgccggctc ggggatgcgg ggggagcgcc ggaccggagc ggagccccgg
     2341 gcggctcgct gctgccccct agcgggggag ggacgtaatt acatccctgg gggctttggg
     2401 ggggggctgt ccccgtgagc tcaataaaag agcccactac aggccggctt gtcgacgacg
     2461 gcggtctccg tcgtcaggat catccgggag tatggccaag cctttgtctc aagaagaatc
     2521 caccctcatt gaaagagcaa cggctacaat caacagcatc cccatctctg aagactacag
     2581 cgtcgccagc gcagctctct ctagcgacgg ccgcatcttc actggtgtca atgtatatca
     2641 ttttactggg ggaccttgtg cagaactcgt ggtgctgggc actgctgctg ctgcggcagc
     2701 tggcaacctg acttgtatcg tcgcgatcgg aaatgagaac aggggcatct tgagcccctg
     2761 cggacggtgc cgacaggtgc ttctcgatct gcatcctggg atcaaagcca tagtgaagga
     2821 cagtgatgga cagccgacgg cagttgggat tcgtgaattg ctgccctctg gttatgtgtg
     2881 ggagggctaa ctcgagcgac gcctcgactg tgccttctag ttgccagcca tctgttgttt
     2941 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     3001 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     3061 tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg
     3121 tgggctctat ccgagcggcc gcgtgttaca accaattaac caattctgat tagaaaaact
     3181 catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt
     3241 gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa
     3301 gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc
     3361 cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg
     3421 agaatggcaa aagcttatgc atttctttcc agacttgttc aacaggccag ccattacgct
     3481 cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga
     3541 ggcgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc
     3601 gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata
     3661 cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac
     3721 ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca
     3781 tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg
     3841 catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag
     3901 cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctggagcaag
     3961 acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca
     4021 gttttattgt tcatgatgat atatttttat cttgtgcaat gtaacatcag agattttgag
     4081 acacaacgtg gctttgttga ataaatcgaa cttttgctga gttgaaggat cagtcatgac
     4141 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     4201 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     4261 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     4321 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     4381 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     4441 agtggctgct gccagtggcg ataagtcgtg tcttaccggg t
//
